CN106456721A - Methods of modulating WARS2 - Google Patents

Abstract

The present invention provides methods and reagents for modulating WARS2 expression, WARS2 activity, or combinations thereof, thereby modulating angiogenesis. Also provided herein are methods for identifying individuals who would benefit from agents that modulate WARS2 expression, WARS2 activity, or combinations thereof.

Description

How to tune WARS2

Background technique

The proliferation of cells is central to the pathophysiology of a variety of diseases. For example, angiogenesis caused by uncontrolled proliferation of endothelial cells plays an important role in, for example, cancer, diabetic eye disease, macular degeneration and arthritis.

There is a need for methods of detecting pathological conditions and treating disorders or diseases associated with uncontrolled proliferation of cells, including, for example, proliferation of endothelial cells that contribute to angiogenesis.

Contents of the invention

As described herein, it has been established that mitochondrial tryptophan tRNA synthetase (WARS2) plays a role in regulating cell proliferation, particularly endothelial cell proliferation, suggesting the benefit of modulating WARS2 in a variety of conditions associated with angiogenesis. As described herein, the present invention provides methods of modulating angiogenesis by controlling WARS2 expression, WARS2 activity, or combinations thereof using a variety of agents including, but not limited to, nucleic acids, small organic molecules, antibodies, aptamers, mutations Body WARS2, or any combination thereof.

In another aspect, the invention provides methods of modulating the proliferation of cells other than endothelial cells, such as cancer cells. Thus, described herein are methods of modulating, for example, cancer cell proliferation by controlling WARS2 expression, WARS2 activity, or combinations thereof using various agents.

Also provided herein are methods of identifying individuals with, eg, tumors or cardiovascular disorders, who would benefit from modulating WARS2 expression, WARS2 activity, or a combination thereof.

Brief description of the drawings

Figures 1A-1C show that genetic mapping of coronary blood flow in the rat genome at three (3) independent time points identifies a single locus on rat chromosome 2q34 that controls coronary blood flow. 1A: Genetic mapping in F2 crosses. Y-axis, confidence of significant association (1=100%, red line=significance threshold); X-axis, position in rat genome. 1B: Use of mapping strains and other strains (BN=Brown Norway, SHR=spontaneously hypertensive rat, LEW=Lewis rat, WKY=Wistar Kyoto rat (Wistar Kyoto haplotype refinement of this region of rat). 1C: Genetic control of gene expression at loci. Two genes were found to be under strong genetic control, Pex11b and Wars2.

Figures 2A-2E show coronary blood flow in various rat strains. (2A) Low blood flow in Lew, SHR and WKY strains, (2B) Rescue of low blood flow in SHR.BN2 congenic. (2C) Different capillary densities in SHR.BN2 congenics. (2D) Genotype-specific effect of genotypes with the SHR allele at the coronary flow (CF) locus, with lower flow. (2E) Compared with the wild-type allele (SEQ ID NO: 8) (high blood flow) in BN rats, in SHR (SEQ ID NO: 5), Lew (SEQ ID NO: 6) and WKY ( In SEQ ID NO:7) (low blood flow), leucine residue 53 was mutated to phenylalanine (F). Shown are mouse (SEQ ID NO: 9), human (SEQ ID NO: 10), chicken (SEQ ID NO: 11), fruit fly (SEQ ID NO: 12), zebrafish (SEQ ID NO: 13) and Returning to the conservation of this residue in yeast (SEQ ID NO: 14).

Figure 3 shows the localization of wild-type (WT) and L53F WARS2 in mitochondria and the impaired enzymatic activity of mutant enzymes. Confocal image analysis of WARS2 cellular localization. Human embryonic kidney cells (HEK293 cells) were transfected with WT or L53F WARS2-GFP, and after 24 hours, cells were fixed, permeabilized and labeled with anti-cox4 (middle two panels). Co-localization of WARS2 with cox4 is shown in the two rightmost panels. Bar size = 5 μm.

Figure 4 shows that WARS2 binds to WARS, an effect that is attenuated by the WARS2(L53F) mutation in the human WARS2 protein; ie WARS2(L53F) binds to WARS less strongly. Human umbilical vein endothelial (HUVEC) cells were transfected with no vector (mock), flag-tagged wild-type WARS2 (WT), or flag-tagged WARS2(L53F) mutant (MUT) and analyzed for the interaction of immunoprecipitated flag proteins with WARS effect.

Figure 5 shows that WARS2 regulates WARS secretion in HUVEC cell culture medium. WARS2 gene silencing using siRNA (top) results in increased WARS secretion in response to IFNγ stimulation. In contrast, WARS2 overexpression (bottom) using an adenoviral vector inhibits WARS secretion.

Figures 6A and 6B: Figure 6A shows that WARS2 silencing (siWARS2) has no effect on spliced WARS transcript expression. Figure 6B shows the subcellular localization of WARS in endothelial cells at baseline and after IFN-γ stimulation.

Figure 7 illustrates that WARS2 binds components of the GAIT complex, an effect not seen in the L53F mutant. HUVEC cells were transfected with Flag-tagged WARS2 or Flag-tagged WARS2(L53F), and the flag-purified fraction was blotted to detect the interaction with GAIT complex proteins.

Figure 8 shows that WARS2 overexpression leads to proteosome degradation of the key GAIT component RPL13a, which can be reversed by addition of the inhibitor MG132. Mutant WARS2(L53F) had no effect on L13a expression.

Figure 9 shows that WARS is secreted from HUVEC into the medium in exosomes, and this effect is not inhibited by brefeldin A (BFA). AnnexA2 = Annexin A.

Figure 10 shows the dose-dependent effect of silencing the zebrafish Wars2 gene using morpholino targeting the Wars2 start codon (ATG MO). This effect was reversed by overexpression of human WARS2 protein.

Figure 11 shows the dose-dependent effects of silencing of the zebrafish Wars2 gene using morpholino targeting the Wars2 start codon on blood flow (upper left), survival (upper right) and cardiac function (lower left). CO = cardiac output; dpf = days post fertilization. Bars, mean; error bars, SEM.

Figure 12 shows abnormal blood vessel formation in Flk-GFP fish inhibited with Wars2, and quantification of dose-dependent effects (lower left).

Figure 13 is an amino acid sequence alignment of rat WARS2 (SEQ ID NO: 1), mouse WARS2 (SEQ ID NO: 2) and human WARS2 (SEQ ID NO: 3). The highlighted sequence "PTGIPHLGNYLGA" shows the position of the mutated lysine residue L in mutant WARS2(L53F).

Figure 14 shows the cDNA sequence of human WARS2 (SEQ ID NO: 4). The highlighted sequences are the regions targeted by the siRNAs.

Figure 15 is a bar graph showing the knockdown of WARS2 mRNA expression in HEK293 cells in the presence of three different WARS2 siRNAs.

Figure 16 is a graph showing that WARS2 SNPs that control WARS2 expression also control WARS levels in human plasma. **, P<0.01.

Figure 17 is a confocal imaging showing that WARS is secreted in exosomes and that expression of WARS2 inhibits the secretion of these exosomes by trapping WARS in cells. HUVEC cells infected with a control virus (Ad. Control, shown in the left panel) or a virus expressing WARS2 (Ad.WRS2, shown in the right panel) were stimulated with interferon γ (Interferonγ, IFN-γ), respectively, and then stained. In control cells, membrane-bound exosomes/microvesicles containing WARS were seen budding from the plasma membrane of the cells (arrows). In contrast, in WARS2-infected cells, a large amount of WARS accumulated at the plasma membrane (*), but not in exosomes.

Figures 18A and 18B show efficient targeting of Wars2 in Brown Norway (BN) rats using zinc finger nucleases. Figure 18A shows genotyping of 8 bp deletions in wild-type BN (Wars2 ^+/+ ) and heterozygous gene-targeted BN (Wars2 ^−/+ ) rats generated using zinc finger nucleases. Lane 1, homozygous wild-type Wars2 ^+/+ ; lanes 2-6, heterozygous Wars2 ^-/+ ; lane 7, markers (50bp, 100bp and 150bp). Figure 18B depicts Wars2 gene expression in BN and F1 (Wars2 ^−/+ ) and F1 (Wars2 ^−/L53F , compound hypomorphic) rats. *, P<0.05; **, P<0.01; ***, P<0.001.

19A-C are rat in vivo data showing zinc finger nuclease-mediated effects on blood vessel density by targeting rat Wars2 using nuclease technology. Figure 19A shows that capillary vessel density is low when Wars2 is low. Figure 19B shows that capillary area is low in the heart when Wars2 is low. Figure 19C illustrates that coronary blood flow dependent on capillary density is low in hearts with low Wars2. Bars, mean; error bars, SEM. **, P<0.01; ***, P<0.001.

Figures 20A and 20B illustrate additional modes of inhibition of WARS2 activity. Figure 20A shows that indoxylin inhibits human mitochondrial WARS2 but not human cytoplasmic WARS activity. Figure 20B shows reduced activity in Wars2 mutant rats (with L53F mutation). **, P<0.01; ***, P<0.001.

Figure 21 shows in vivo knockdown of Wars2 in the eye using cholesterol-modified siRNA. "NT" indicates non-targeting siRNA. Data are the mean of results from three rats. **, P<0.01.

Figures 22A-F illustrate the role of WARS2 in an in vitro model of angiogenesis. Figures 22A-C show loss of function of WARS2 as evidenced by reduced EC tube formation; Figures 22D-F show gain of function of WARS2 using adenovirus (Ad)-mediated overexpression of WARS2 compared to GFP. Total EC tube (Figures 22A and 22D), total EC tube length (Figures 22B and 22E) and total branch points (Figures 22C and 22F). Bars, mean; error bars, SD. ***, P<0.001.

Figures 23A and 23B show the effect of WARS2 inhibition on EC morphology and cell number. Figure 23A shows super-resolution microscopy of ECs transfected with siNT or siWARS2 and stained for actin (left), mitochondria (middle) and composite image with nuclear staining (right) (scale bar = 25 μm). In the presence of siWARS2, essentially all actin stress fibers disappeared, cell edges took on a wrinkled appearance (left and right panels), and mitochondria became clumpy (middle and right panels). Figure 23B shows EC numbers in cultures transfected with siNT or siWARS2 for 72 hours. Bars, mean; error bars, SD. *, P<0.01, ***, P<0.001.

Figures 24A and 24B illustrate the effect of WARS2 inhibition on the cell cycle. Figure 24A shows that siRNA-mediated inhibition of WARS2 produces multiple cells with two or more incompletely separated nuclei in endothelial cells (EC). Nuclei (wedges); (bar = 50 μm). Figure 24B is a FACS analysis of the cell cycle in proliferating EC treated with non-targeting siRNA (siNT) or siRNA against WARS2 (siWARS2); results show a significant reduction in EC in S phase, as well as the number of G2/M phases treated with siWARS2 increase (see 4N increase from 19.62% to 30.81%). The experiment was repeated (n=3) with similar results.

Figure 25 illustrates that inhibition of Wars2 prevents blood vessel growth in a model of breast cancer angiogenesis. Arrows indicate areas of vessel growth into vascular endothelial growth factor (VEGF) matrigel plugs from animals expressing normal Wars2. In contrast, virtually no growth into the plug was seen from animals carrying the Wars2(L53F) mutation.

Figure 26 shows that cell proliferation of the A549 lung cancer cell line can be reduced by reducing the expression of WARS2. Data are the mean of three experiments; standard deviation bars are shown. "***" indicates significant at P<0.005.

Detailed description of the invention

The following is a description of exemplary embodiments of the present invention.

Genetic determinants of coronary blood flow were mapped and rat chromosomes that genetically control blood flow in the heart were identified in an experimental rat cross (Brown Norway x Spontaneously Hypertensive Rat F2 Population) as described herein loci on 2. This effect was confirmed in congenic rats that had reduced capillary density. Haplotype mapping and eQTL analysis prioritized tryptophan tRNA synthetase 2 (Wars2; mitochondrial localization) as a candidate gene at the locus, and sequencing of Wars2 identified the L53F mutation in the highly conserved ATP-binding domain (See Figure 13).

Aminoacyl tRNA synthetases can form heterodimeric complexes, and WARS2 was observed to bind WARS, thereby inhibiting VEGFA signaling and angiogenesis when cleaved and secreted. Binding of WARS2 to WARS is largely abolished in the L53F mutant form. In IFN-γ-stimulated endothelial cells, WARS2 overexpression limited the secretion of antiangiogenic WARS fragments, whereas WARS2 knockdown increased their release. WARS exhibits atypical secretion and, as demonstrated herein, WARS is secreted in exosomes. While WARS is predominantly localized in the cytoplasm, increased WARS was found in the mitochondrial compartment, and decreased intracellular retention and cleavage of WARS was observed in the presence of WARS2 overexpression. In a zebrafish model, morpholino-mediated inhibition of Wars2 resulted in reduced vascular lumenization, disruption of arterial architecture, and impaired cardiac function. Furthermore, as described herein, inhibition of WARS2 activity or expression results in decreased endothelial cell proliferation.

Taken together, the data herein identify WARS2 as a novel gene for the regulation of angiogenesis. Indeed, the WARS2 enzyme active site and binding to WARS affect angiogenesis. As shown herein, targeting WARS2 biologically regulates the secretion of extracellular vesicles (e.g., exosomes) of WARS, as well as the proliferation of endothelial cells, and this effect can be used to suppress abnormal blood vessels in individual (e.g., human) diseases occur, diseases such as cancer, especially breast cancer in which WARS2 is involved, eye diseases and metabolic disorders such as obesity (where WARS2 mutations are implicated in a role in these diseases). The data herein demonstrate that mutations in WARS2 can have profound effects on WARS function, endothelial cell proliferation, and thus angiogenesis.

In another aspect, the data presented herein show that inhibition of WARS2 also results in a significant reduction in cancer cell numbers, suggesting a broader role in regulating cell proliferation beyond endothelial cells, including, for example, regulating the proliferation of cancer cells themselves.

As used herein, the term "WARS2" refers to mitochondrial aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNAs to their cognate amino acids. Specifically, "WARS2" is a mitochondrial tryptophan tRNA synthetase. Two forms of tryptophan-tRNA synthetase exist, the cytoplasmic form known as WARS and the mitochondrial form known as WARS2.

As used herein, for example, the human form of WARS2 is in all capital letters, where the DNA/RNA form is italicized (WARS2) and the protein form is not italicized (WARS2). Typically, non-human forms (eg, mouse or rat) are indicated as Wars2 in lowercase except for the first letter, where the DNA/RNA form is italicized (Wars2) and the protein form is not italicized (Wars2).

Accordingly, in one aspect, the invention relates to a method of modulating angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that modulates WARS2 expression, WARS2 activity, or a combination thereof.

As used herein, "angiogenesis" refers to the proliferation of endothelial cells that form blood vessels.

As used herein, the term "modulating" angiogenesis refers to altering angiogenesis. For example, methods of modulating angiogenesis include inhibiting, enhancing or maintaining angiogenesis. Accordingly, in another aspect, the invention relates to a method of inhibiting angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof.

In some aspects, methods of inhibiting angiogenesis are useful for inhibiting angiogenesis caused by or occurring as a result of one or more conditions or diseases. For example, the methods can be used to inhibit retinopathy (e.g., diabetic retinopathy) or macular degeneration (e.g., age-related macular degeneration (age-related macular degeneration, AMD), such as wet AMD or dry AMD) in an individual of angiogenesis. Alternatively, methods of inhibiting angiogenesis can be used to inhibit angiogenesis in cancer (eg, inhibit the growth of blood vessels that support tumor growth) (Curtis, C. et al., Nature, 486:346-352 (2012); Aberg, UWN et al., PLoS One, 6e:25720 (2011); Damasceno M et al Curr Opin Oncol, 23 Suppl:S3-9 (2011); Huana C-C et al, PLoS One, 8e:76421 (2013)) or metabolic disorders such as obesity (Heid et al, Nature Genetics , 42:949-960 (2010); Rosen ED et al., Cell, 156:20-44 (2014); Koza, RA et al., J Biol Chem, 275:34486-34492 (2000)).

In other aspects, the invention relates to methods of treating retinopathy in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof. In a specific aspect, the retinopathy is diabetic retinopathy.

In other aspects, the invention relates to a method of treating macular degeneration (eg, wet AMD; dry AMD) in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof.

In other aspects, the invention relates to methods of enhancing angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that increases WARS2 expression, WARS2 activity, or a combination thereof.

In some aspects, methods of enhancing angiogenesis are useful for enhancing angiogenesis in individuals undergoing and/or having undergone transplantation (eg, organ transplantation) or wound healing, or exhibiting various forms of cardiovascular disease.

As used herein, "cardiovascular disease" includes, but is not limited to, angina, myocardial infarction, heart failure, atherosclerosis, and angiosarcoma.

In another aspect, modulation of WARS2, as described herein, can modulate the proliferation (growth) of cells other than endothelial cells (eg, cancer cells). In particular, inhibiting WARS2 expression or WARS2 activity can reduce the number of cancer cells. Accordingly, the present invention relates to methods of modulating cancer cell proliferation in an individual in need thereof comprising administering an effective amount of an agent that modulates WARS2 expression, WARS2 activity, or a combination thereof. As used herein, "cancer cell proliferation" refers to the proliferation of cancer cells themselves.

As used herein, the term "modulating" cancer cell proliferation refers to altering the ability of cancer cells to proliferate. For example, methods of modulating cancer cell proliferation include inhibiting, enhancing or maintaining cancer cell proliferation.

In a specific aspect, the invention relates to a method of inhibiting cancer cell proliferation in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof.

As described herein, the agents used in the methods modulate (eg, inhibit, enhance) the expression of WARS2, the activity of WARS2, or a combination thereof. The activity of WARS2 includes the interaction (eg, binding) of WARS2 with one or more partners. For example, as shown herein, WARS2 interacts with proteins of the WARS and Gamma-interferon Activated Inhibitor of Translation (GAIT) complex. Specific examples of proteins of the GAIT complex that interact with WARS2 include glutamyl-prolyl tRNA synthetase (EPRS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ribosomal protein L13a (RPL13a). Thus, in some aspects, the reagents used in the methods modulate the interaction of WARS2 with WARS, EPRS, GAPDH, RPL13a, or combinations thereof.

Any of a variety of reagents can be used in the methods provided herein. For example, the agent can be a nucleic acid (eg, DNA, RNA), a small organic molecule, an antibody, an aptamer, a mutant WARS2, or a combination thereof. Such agents can readily be tested, eg, according to the methods exemplified herein, to assess whether they have the desired effect (eg, inhibition or activation/enhancement) on WARS2 expression, WARS2 activity, or a combination thereof.

In some specific aspects, the agent is a nucleic acid that binds to all or a portion of a nucleic acid that encodes WARS2, modulates WARS2 expression, encodes all or a portion of a polypeptide that interacts (e.g., binds) with WARS2, and/or modulates interaction with WARS2 Expression of the active polypeptide. Examples of such nucleic acids that regulate the expression of polypeptides that interact with WARS2 or the expression of WARS2 itself include small interfering ribonucleic acid (siRNA), antisense, short hairpin RNA (short hairpin RNA, shRNA), appropriate Ligands and/or morpholino oligomers. In other aspects, the agent is a vector that directs the expression of WARS2 and/or a polypeptide that interacts with (eg, binds to) WARS2. In one aspect, the agent is a vector directing overexpression of WARS2. Other examples of inhibitory nucleic acids suitable for use in the invention and methods of designing such nucleic acids are readily available to those skilled in the art. See, eg, US Patent Application No. 20150017091. In a specific aspect, as exemplified herein, the agent is an siRNA that reduces or inhibits the expression of WARS2. In one aspect, the siRNA pair against WARS2 is SEQ ID NO: 50 and 51; SEQ ID NO: 52 and 53; or SEQ ID NO: 54 and 55. In another aspect, the siRNA pair against Wars2 is SEQ ID NO:48 and 49.

In other aspects, the agent is a small organic molecule that directly or indirectly affects WARS2 expression, WARS2 activity, or a combination thereof. Small organic molecules that can modulate WARS2 expression and/or WARS2 activity can be achieved using standard methods and as described in any of the assays exemplified herein. An agent modulates WARS2 expression and/or WARS2 activity if WARS2 expression and/or WARS2 activity is altered in the presence of the agent compared to a control. In one aspect, the agent is indomycin or any effective derivative thereof.

In other aspects, the agent is an antibody that directly or indirectly affects WARS2 expression, WARS2 activity, or a combination thereof. Methods for identifying, isolating and producing antibodies and active fragments thereof against any suitable target are well established and known in the art. Unless the context indicates otherwise, the term "antibody" is used in the broadest sense and specifically covers antibodies (including full-length monoclonal antibodies) and antibody fragments so long as they recognize and bind WARS2. Antibody molecules are usually monospecific, but can also be described as heterospecific or multispecific. Antibody molecules bind to specific antigenic determinants or epitopes on antigens through specific binding sites. An "antibody fragment" comprises a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; Thus, in one aspect, the agent is an antibody that binds to WARS2 and modulates its activity by, for example, modulating its binding to WARS. In other aspects, antibodies can target and bind to proteins that regulate WARS2 expression, thereby indirectly modulating WARS2. Methods for screening the effect of antibodies on WARS2 expression or WARS2 activity can be performed using standard methods and as described in any of the assays exemplified herein.

Methods for preparing polyclonal antibodies are readily available to those skilled in the art. See, for example, Green, et al., Production of Polyclonal Antisera in: Immunochemical Protocols (Manson, ed.), pp. 1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1 (1992). Methods for preparing monoclonal antibodies are also available to those skilled in the art. See, eg, Kohler & Milstein, Nature, 256:495 (1975); Coligan, et al., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub.( 1988)), which is incorporated herein by reference. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such separation techniques include affinity chromatography with protein-ASepharose, size exclusion chromatography and ion exchange chromatography. See, eg, Coligan, et al., Sections 2.7.1-2.7.12 and 2.9.1-2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG), In: Methods in Molecular Biology, Vol. 10, 79-104 Page (HumanaPress (1992). Methods for in vitro and in vivo manipulation of monoclonal antibodies are also available to those skilled in the art. For example, monoclonal antibodies for use in accordance with the present invention may be described first by Kohler and Milstein, Nature 256, 495 (1975). prepared by the hybridoma method, or can be prepared by recombinant methods, for example, the method described in U.S. Pat. etc., the technology described in J.Mol.Biol.222:581-597 (1991), is isolated from phage antibody library.In addition, humanized monoclonal antibody is to produce containing people's specificity and recognizable sequence by recombinant method Antibodies are well known. See reviews Holmes et al., J. Immunol., 158:2192-2201 (1997) and Vaswani et al., Annals Allergy, Asthma & Immunol., 81:105-115 (1998).

In another aspect, the agent is an aptamer that directly or indirectly affects WARS2 expression, WARS2 activity, or a combination thereof. "Aptamer" refers to a nucleic acid molecule capable of binding a particular molecule of interest with high affinity and specificity. Aptamers are typically developed to bind specific ligands by using a known in vivo or in vitro selection technique known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Methods for preparing aptamers are described, for example, in Ellington and Szostak, Nature 346:818 (1990), Tuerk and Gold, Science 249:505 (1990), U.S. Patent No. 5,582,981, PCT Publication No. WO 00/20040, U.S. Patent No. 5,270,163, Lorsch and Szostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry 36:9726 (1997), Blind, Proc.Nat'l.Acad.Sci.USA 96:3606-3610 (1999), Huizenga and Szostak, Biochemistry, 34:656-665 (1995), PCT Publication Nos. WO 99/54506, WO 99/27133, WO 97/42317 and US Patent No. 5,756,291. When appropriate in the present invention, aptamers can be used to activate or inhibit the expression of WARS2, or directly bind to WARS2 to regulate its activity. Methods for screening and designing aptamers are well established and readily available to those skilled in the art.

In other aspects, the agent is a mutant WARS2 that affects WARS2 expression, WARS2 activity, or a combination thereof. For example, as described herein, WARS2 mutants with the L53F substitution modulate wild-type WARS2 binding to WARS. Thus, if inhibition of WARS2 activity is desired (e.g., to inhibit endothelial cell proliferation, thereby inhibiting angiogenesis; or to inhibit cancer cell proliferation), vectors carrying the L53F mutant form of WARS2 can be targeted in vivo using known gene delivery methods (see For example, US Patent No. 8,741,279 and Ramamoorth M and Narvekar A. Non-viral vectors in genetherapy-an overview. J. Clin Diagn Res. 2015 Jan;9(1):GE01-6.doi:10.7860/JCDR/2015 /10443.5394.Epub Jan 1, 2015). In another aspect, WARS2 expression in tumors can be completely disrupted in vivo using known methods of targeted gene disruption.

Without wishing to be bound by any one particular mechanism of action, the data described herein suggest that WARS2 acts as a regulator of endothelial cell proliferation (and thus angiogenesis) through one or more mechanisms. For example, inhibition of WARS2 resulted in a dramatic loss of stress fibers required for endothelial cell migration and division. The absence of fibers can inhibit and/or preclude endothelial cell proliferation and blood vessel formation (angiogenesis); there is incomplete nuclear separation and accumulation of cells in G2/M phase is seen. The inability to divide eventually leads to cell death. Similarly, WARS2 may act as a regulator of proliferation of other cell types such as cancer cells due to incomplete cell division.

Furthermore, WARS2 may function by adversely affecting energy production in mitochondria. Oxygen consumption was significantly reduced when WARS2 was inhibited. The loss of energy required for cell migration and division may similarly result in the inability of cells to divide, arrest in G2/M phase, and eventual death. In both cases, the result is inhibition of angiogenesis in the event of failure of endothelial cells to divide and proliferate to form blood vessels. The data herein demonstrate a significant reduction in endothelial cell tube formation in the presence of inhibited WARS2. In the case of cancer cells, the result is cell death and a reduction in cancer cell proliferation.

Again, without wishing to be bound by any particular mechanism of action, additional data described herein suggest that WARS2 acts as a regulator of angiogenesis by interacting with exosomes and inhibiting secretion of WARS from exosomes. In the experiments described herein, gain-of-function of WARS2 reduced WARS secretion. Reduction of WARS secretion is expected to have downstream effects on angiogenesis.

Accordingly, in other aspects, the invention relates to methods of identifying the angiogenic potential of a tumor in an individual in need thereof. As used herein, the "angiogenic potential" of a tumor refers to the body's ability to stimulate tumor growth due to the proliferation of blood vessels that infiltrate the tumor and, for example, provide nutrients and oxygen to the tumor, and/or remove waste products from the tumor. In a specific aspect, the angiogenic potential of a tumor refers to the ability of the tumor to invade (invasive potential) and/or metastasize (metastatic potential) into tissues (eg, tissues near or distant from the tumor location).

In one aspect, the method comprises determining one or more extracellular vesicles (extracellular vesicles, EVs) (eg, one or more exosomes) and/or their contents obtained from an individual compared to a control. WARS levels in animals. An increase in the level of WARS in one or more EVs and/or their contents compared to controls indicates proliferation of blood vessels that penetrate into the tumor (e.g., provide nutrients and oxygen thereto and/or remove waste products therefrom) The likelihood is low, therefore, the tumor in said individual has a low angiogenic potential. A decrease in the level of WARS in one or more EVs and/or their contents compared to a control indicates proliferation of blood vessels that penetrate into the tumor (e.g., provide nutrients and oxygen thereto and/or remove waste products therefrom) The likelihood is high that, therefore, the tumor in said individual has a high angiogenic potential. The method may also include obtaining one or more EVs and/or their contents from the individual.

In a particular aspect, a method of identifying the angiogenic potential of a tumor in an individual in need thereof comprises obtaining one or more EVs (e.g., exosomes) and/or their contents from the individual; and determining the relative WARS levels in the one or more EVs and/or their contents compared to a control. An increase in the level of WARS in one or more EVs and/or their contents compared to controls indicates a lower likelihood of proliferation of blood vessels infiltrating the tumor and, therefore, a tumor with low angiogenic potential in said individual . A reduction in the level of WARS in one or more EVs and/or their contents compared to controls indicates a higher likelihood of proliferation of blood vessels infiltrating the tumor and, therefore, a tumor with high angiogenic potential in said individual .

As used herein, an extracellular vesicle (EV) is a vesicle that is released (eg, secreted) by a cell(s). In some specific aspects, EVs are exosomes, which are membrane vesicles of endocytic origin of approximately 30-100 nm, secreted by most cell types (eg, in vivo; in vitro). In other aspects, EVs are exosomes, which are membrane vesicles of endocytic origin of approximately 100-1000 nm, secreted by most cell types (eg, in vivo; in vitro). EVs are also found in vivo in body fluids (eg, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk) and tissues. Methods of obtaining and analyzing EVs and the contents of one or more EVs are known to those skilled in the art, see, e.g., Wang et al., J Biol Chem, published online September 3, 2013, which is incorporated in its entirety This article. In some particular aspects, the one or more EVs are isolated, purified, substantially purified, and/or partially purified.

The method of identifying the angiogenic potential of a tumor may further comprise obtaining a sample (biological sample) comprising one or more EVs. A sample can be obtained from a biological fluid, biological tissue, or a combination thereof. Examples of biological samples used in this method include biological fluids (eg blood, serum, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, milk) and biological tissues (eg organ tissue, tumor tissue) and the like.

The method of identifying the angiogenic potential of a tumor can also include treating an individual based on the angiogenic potential of the tumor. For example, the method may also include treating an individual with a tumor having a high angiogenic potential with an anti-angiogenic therapy and/or a therapy in addition to an anti-angiogenic therapy. Alternatively, the method may also include treating an individual with a tumor with low angiogenic potential with a therapy in addition to an anti-angiogenic therapy (e.g., a chemotherapeutic agent that does not inhibit angiogenesis), or with an anti-angiogenic therapy in combination with The individual is treated with a therapy other than an anti-angiogenic therapy. In certain aspects, if the level of WARS in the one or more EVs and/or their contents is reduced compared to a control, treating the EVS with an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof described individual. It will be apparent to those skilled in the art that a variety of controls are available in the method of identifying the angiogenic potential of a tumor. In one aspect, the control is the level of WARS in one or more EVs obtained from one or more tumor-free individuals (e.g., in one or more EVs comprising EVs from tumor-free individuals, such as one or more healthy individuals). in more samples).

In another aspect of the invention, a method of identifying angiogenic potential in an individual suffering from a cardiovascular disorder is provided. As used herein, "angiogenic potential" of a cardiovascular disorder refers to the ability of the body to modulate (ie, increase or decrease) the cardiovascular response due to proliferation of blood vessels that provide coronary blood flow to the heart.

In one aspect, the method comprises determining one or more extracellular vesicles (EVs) (e.g., one or more exosomes) and/or their WARS levels in inclusions. An increase in the level of WARS in one or more EVs and/or their contents compared to a control indicates low or reduced angiogenic potential in said individual and thus indicates low coronary blood flow into the heart, This can lead to, for example, angina and heart failure. A reduction in the level of WARS in one or more EVs and/or their contents is indicative of a high or increased angiogenic potential in said individual compared to a control, which can lead to, for example, increased atherosclerosis. In certain aspects, high or increased angiogenic potential is associated with obesity as a result of adipocyte growth. The method may also include obtaining one or more EVs and/or their contents from the individual.

In a particular aspect, a method of identifying angiogenic potential in an individual with a cardiovascular disorder comprises obtaining one or more EVs (e.g., exosomes) and/or their contents from said individual; WARS levels in the one or more EVs and/or their contents compared. The control can be the level of WARS in one or more EVs obtained from one or more individuals without a cardiovascular disorder.

The method of identifying angiogenic potential in an individual with a cardiovascular disorder may also comprise obtaining a sample (biological sample) comprising one or more EVs. A sample can be obtained from a biological fluid, biological tissue, or a combination thereof. Examples of biological samples used in this method include biological fluids (eg blood, serum, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, milk) and biological tissues (eg organ tissue, tumor tissue) and the like.

The method of identifying angiogenic potential in an individual with a cardiovascular disorder can also include treating the individual based on the identified angiogenic potential of the individual with a cardiovascular disorder. For example, the method may also include treating an individual with a cardiovascular disorder having low or reduced angiogenic potential compared to a control with a suitable therapeutic agent to reduce or prevent angina or heart failure. In another example, the method can further comprise treating an individual with a cardiovascular disorder having a high or increased angiogenic potential compared to a control with a suitable therapeutic agent to reduce or prevent the effects of atherosclerosis. In certain aspects, if the level of WARS is increased in one or more EVs and/or their contents compared to a control, the individual is treated with an effective amount of an agent that enhances WARS2 expression, WARS2 activity, or a combination thereof to treat Examples include angina or heart failure. In other aspects, if the level of WARS is reduced in one or more EVs and/or their contents compared to a control, the individual is treated with an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof to treat e.g. Effects of atherosclerosis.

In other aspects, the invention relates to methods of determining whether an individual in need thereof would benefit from anti-angiogenic therapy. The method comprises performing genomic analysis on all or part of chromosome 1 of the individual, wherein the part comprises

TTCATATTCTGTCGAGACACCCATC[C/G]CCCTGTGTTTCACTTGTCTGATTAC (SEQ ID NO:38), and at least one allele at position 26 of SEQ ID NO:38 was detected. If at least one allele is G at position 26 of SEQ ID NO: 38, the individual will benefit from anti-angiogenic therapy. In some aspects, one allele at position 26 of SEQ ID NO: 38 in the individual is G and the other allele is C (the individual is heterozygous for the allele at position 26, G : C). In other aspects, both alleles at position 26 of SEQ ID NO: 38 in the individual are G (the individual is homozygous for the allele at position 26, G:G). In other aspects, both alleles at position 26 of SEQ ID NO: 38 in the individual are C (the individual is homozygous for the allele at position 26, C:C). In a specific aspect, said individual has a tumor.

The portion of chromosome 1 comprising SEQ ID NO: 38 comprises positions 119,503,593-119,504,093 of chromosome 1 based on the Human February 2009 (GRCh37/hg19) collection of Homo sapiens generated by the Genome Reference Consortium.

A variety of genomic analysis methods are available in methods of determining whether an individual will benefit from anti-angiogenic therapy. Examples of such methods include sequencing analysis (next generation sequencing), electrophoresis (gel, capillary, temperature gradient gel electrophoresis), polymerase chain reaction (high resolution melting analysis), mass spectrometry, single-strand conformation polymorphism (single strand conformation polymorphism, SSCP), electrochemical analysis (using ferrocenyl naphthalimide), high performance liquid chromatography (high performance liquid chromatography, HPLC) (denaturing HPLC), enzyme analysis (restriction fragment length more Restriction fragment length polymorphism (RFLP), flap endonuclease (flapendonuclease), primer extension, 5′-nuclease, oligonucleotide ligation), hybridization analysis (dynamic allele-specific hybridization, allele Gene-specific oligonucleotide (allele-specific oligonucleotide (aso) analysis, molecular beacon, microarray (SNP microarray)), mismatch binding protein, or a combination thereof.

The method of determining whether an individual in need thereof would benefit from anti-angiogenic therapy may further comprise comparing the detection of at least one allele at position 26 of SEQ ID NO: 38 from said individual to a control. Any suitable control may be used, including alleles present in one or more individuals who respond to anti-angiogenic therapy, who do not respond to anti-angiogenic therapy, or combinations thereof.

The method of determining whether an individual would benefit from anti-angiogenic therapy may also comprise obtaining a sample (biological sample) from said individual. The sample can be obtained from biological fluid, biological tissue, or a combination thereof. Examples of biological samples used in the method include biological fluids (eg blood, serum, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, milk) and biological tissues (eg organ tissue, tumor tissue) and the like.

The method of determining whether an individual would benefit from anti-angiogenic therapy can also include treating an individual having at least one allele that is G at position 26 of SEQ ID NO:38 with an anti-angiogenic therapy. Various anti-angiogenic treatments are known to those skilled in the art, including inhibition of vascular endothelial growth factor (vascular endothelial growth factor, VEGF), VEGF receptor-2 (VEGF receptor-2, VEGFR), platelet derived growth factor (platelet derived growth factor) factor, PDGF), Tie-1 receptor (Tie-1 receptor, Tie-1R), Tie-2 receptor (Tie-2 receptor, Tie-2R), placental growth factor (placental growth factor, PlGF), or a combination thereof.

As used herein, "individual" refers to an animal, and in one particular aspect a mammal. Examples of mammals include primates, dogs, cats, rodents, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice.

The term "subject in need thereof" refers to a subject in need of treatment or prophylaxis, as determined by a researcher, veterinarian, physician or other clinician. In one embodiment, the individual in need thereof is a mammal, such as a human.

The need or desire for administration according to the methods of the invention is determined by using known risk factors. In the final analysis, the effective amount of the particular agent(s) (e.g., composition) is determined by the physician in charge of the case, but depends on factors such as the exact condition and/or disease to be treated, the patient's The severity of the condition and/or disease, the route of administration chosen, other medications and treatments the patient may concomitantly require, and other factors in the physician's judgment.

An effective amount of an agent that modulates WARS2 is administered to an individual in need thereof. As used herein, "effective amount" or "therapeutically effective amount" means the amount of the active composition that will elicit a desired biological or medical response in a tissue, system, subject or human, including alleviation of the condition being treated and and/or all or some of the symptoms of a disease.

Compositions can be administered in a single dose (eg, throughout the day) or in multiple doses. Furthermore, the compositions can be administered over one or more days (eg, consecutive or non-consecutive days).

Nucleic acids that modulate WARS2 expression and/or WARS2 activity can be delivered to cells in vitro and in vivo by a variety of methods known to those of skill in the art, including direct contact with cells (e.g., "naked" siRNA) or by interaction with one or more Combinations of agents that facilitate targeting or delivery into cells. Such agents and methods include lipoplexes, liposomes, iontophoresis, hydrogels, cyclodextrins, nanocapsules, microspheres and nanospheres, and protein carriers (e.g., Bioconjugate Chem. (1999 ) 10:1068-1074 and WO 00/53722). The nucleic acid/vehicle combination can be delivered locally in vivo by direct injection or by use of an infusion pump. Nucleic acids can be delivered in vivo by a variety of means including intravenous, subcutaneous, intramuscular or intradermal injection or inhalation.

The use of compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified or long-circulating liposomes or stealth liposomes) is also known. These formulations provide a means of increasing the stability of liposomes or lipoplexes in solution by preventing their aggregation and fusion. The formulation also has the added benefit of resistance to opsonization in vivo and elimination through the mononuclear phagocytic system (MPS or RES), resulting in longer blood circulation time and enhanced tissue exposure of the encapsulated drug . Such liposomes have also been shown to accumulate selectively in tumors (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238, 86-90). Long-circulating liposomes enhance DNA and RNA pharmacokinetics and pharmacodynamics. Long-circulating liposomes also protect siRNA from nuclease degradation.

Nucleic acid agents that modulate WARS2 expression and/or WARS2 activity can be formulated as pharmaceutical compositions. The pharmaceutical composition can be used as a medicine or a diagnostic agent alone or in combination with other agents. For example, one or more siRNAs of the invention can be combined with a delivery vehicle (eg, liposome) and excipients (eg, carrier, diluent). Lipid-mediated nucleic acid delivery methods are well known (see eg US Patent Application No. 20150017091). Other agents such as preservatives and stabilizers may also be added. Methods for delivering nucleic acid molecules are known in the art, and are for example described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol . Memb. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, USA Described in Patent No. 6,395,713 and PCT WO 94/02595. Nucleic acids may also be administered in combination (separately or simultaneously) with other therapeutic compounds, eg, as a combined unit dose. In one embodiment, the invention comprises one or more nucleic acids according to the invention (e.g. siRNA) pharmaceutical composition.

A pharmaceutical composition comprising an agent that modulates WARS2 expression and/or WARS2 activity can be formulated together with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carriers and compositions can be sterile. The formulation should suit the mode of administration.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions (such as NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, Amylose or starch, glucose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof. The pharmaceutical preparations can, if desired, be combined with adjuvants which do not deleteriously react with the active compounds, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffers, colorants, flavors and/or aroma substances etc. mixed.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

Agents that modulate WARS2 expression and/or WARS2 activity may also be administered as part of combination therapy with other compounds (e.g., vascular endothelial growth factor (VEGF); VEGF inhibitors such as WARS, VEGF receptor-2 (VEGFR), platelet-derived Growth factor (PDGF), Tie-1 receptor (Tie-1R), Tie-2 receptor (Tie-2R), YARS1 (micro-Yars1) (Ewalt and Schimmel, Biochem, 41(45): 13344-13349 (2002 )); placental growth factor (PlGF), or a combination thereof).

The reagents can be formulated into pharmaceutical compositions suitable for human administration according to conventional procedures. For example, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. The composition may also contain a solubilizer and a local anesthetic to relieve pain at the injection site, if desired. Generally, the ingredients are presented alone or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active compound. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Compounds are administered in therapeutically effective amounts. The amount of compound that is therapeutically effective in treating a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The precise dosage employed in the formulation will also depend on the route of administration and the severity of the symptoms of angiogenic, vascular, cardiac or circulatory disease, and should be decided according to the judgment of the physician and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In another aspect, the invention relates to methods of identifying agents that modulate WARS expression and/or WARS activity. The method comprises determining WARS expression and/or WARS activity in one or more EVs (e.g., exosomes) obtained from cells, tissues, and/or organisms that have been contacted with an agent to be assessed; The WARS expression and/or WARS activity is compared to a control, wherein the agent modulates WARS expression and/or WARS expression and/or WARS activity if the WARS expression and/or WARS activity is altered in the presence of the agent as compared to the control active. The method may also include contacting the cell, tissue and/or organism with an agent to be assessed. The method may further comprise obtaining one or more EVs from cells, tissues and/or organisms that have been contacted with the agent to be assessed.

In a particular aspect, the invention relates to a method of identifying an agent that modulates WARS expression and/or activity, comprising making a cell(s), tissue and/or organism (which comprises one or more EVs such as exogenous exosomes) are contacted with the reagent to be assessed; one or more EVs are obtained from the cell, tissue and/or organism after contact with the reagent; and/or WARS expression and/or activity in one or more EVs obtained from an organism; and comparing said WARS expression and/or activity with a control. The agent modulates WARS expression and/or activity if WARS expression and/or activity is altered in the presence of the agent as compared to a control.

Determination of WARS expression by EVs includes determining the amount of WARS expressed within or secreted from EVs. Determination of WARS activity of EVs includes determining whether WARS is secreted from EVs, and whether or to what extent WARS interacts with (eg, binds to; associates with) WARS2.

In some aspects, WARS expression and/or activity of EVs is reduced following contact with the agent as compared to a control. In other aspects, WARS expression and/or activity of EVs is increased following contact with the agent as compared to a control.

It will be apparent to those skilled in the art that a variety of controls can be used in the methods of the invention. One example of a suitable control is WARS expression and/or activity in one or more EVs obtained from cells, tissues and/or organisms that have not been contacted with the agent.

In some aspects, the cells, tissues and/or organisms contacted with the agent to be assessed can be from the same individual. In other aspects, cells, tissues and/or organisms can be from different individuals of the same species. In other aspects, the cells, tissues and/or organisms can be from different species.

Example

Example 1

Identification of WARS2 as a novel gene for coronary blood flow and capillary density

method

Rat parent strain

Four parental strains were studied: spontaneously hypertensive rats (SHR) were included as hypertensive strains, while Brown Norway (BN), Wistar Kyoto (WKY) and Lewis were included as normotensive control strains. Male rats aged 12 to 14 weeks were phenotyped at a rate of two animals/day. All rats were purchased from Charles River UK Limited (Margate, UK).

BN-SHR F2 group

Rats are bred through a monogamous mating system. BN females were crossed with SHR males to produce BN x SHR(BXH)Fl animals, and reciprocal crosses were performed to obtain SHR x BN(HXB) animals. F1 BXH animals were crossed to generate F2BXH animals, and F1 HXB animals were crossed to generate F2 HXB animals. Animals were maintained at the Central Biomedical Service Facility at Imperial College London and housed up to 5 per cage. Animals have random access to standard rat chow and sterile water. Except for breeding purposes, animals are separated according to sex. They maintain a 12-hour daily cycle with automatic light switching. Colonies of specific pathogens were tested regularly by using sentinels kept in individual cages. All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 (UK Animals (Scientific Procedures) Act).

blood pressure (BP)

BP was measured using a carotid artery cannula in each animal prior to cardiac resection for ex vivo phenotyping. An ultra-miniature 2mm pressure catheter (MPVS-Ultra Single Segment Foundation System, ADI Instruments) was used. Calibrate the pressure catheter against an external manometer prior to measurement. Animals were then anesthetized using inhaled 4% isoflurane. The carotid region is exposed, an arteriotomy is performed and the catheter is placed in place. Data were then captured continuously by LabchartPro software (ADI Instruments) and analyzed offline. Following carotid cannulation, the concentration of inhaled isoflurane was gradually reduced from 4% to 1.5% to allow BP to reach physiologic levels. After recording BP data, the pressure catheter was removed from the vessel and then tied off before CF measurement.

Coronary flow (CF)

CF was measured using the Langendorff formulation. Immediately after resection, hearts were placed in ice-cold Kreb buffer and transferred to an ex vivo perfusion device. Clamp the aorta to the cannula via an arterial clip and secure using 3/0 silk. Hearts were perfused with modified Krebs-Henseleit buffer, prepared as suggested by Sutherland and Hearse. The solution was kept in a jacketed reservoir at 37°C and continuously gassed with carbogen solution (95% oxygen, 5% _CO2 ) to maintain a pH of 7.35-7.45. Perfusion was established no more than three minutes after cardiotomy. The left atrium (LA) was removed, and a fluid-filled latex ball was placed in the left ventricular (LV) cavity. LV systolic (LV dP/dtmax) and LV diastolic (dP/dtmin) are derived from LV pressure. Coaxial bipolar electrodes were placed on the right atrium and pacing was initiated at 360 beats per minute. Electrodes are placed over the right atrium and left ventricle to record a continuous ECG. The entire preparation was kept in a vessel maintained at 37°C. The perfusate was pumped to the heart at a constant volume with a perfusion pressure of 90 mmHg. CF was measured by an in-line ultrasonic flow meter placed in a component close to the ventricle, with a bubble trap to prevent cardiac air embolism. Hemodynamic data were continuously captured by LabchartPro software (ADI Instruments) and analyzed offline after the experiment. Isolated heart preparations were studied for 15 min at baseline. This was followed by a brief one minute global ischemia and reperfusion to assess maximal hyperemia. Myocardial infarction was subsequently induced for 35 min by ligation of the proximal LAD, followed by one hour of reperfusion. Cardiac eluates were collected at baseline and during the first 10 min of reperfusion to measure metabolite levels.

gene expression data

For a subgroup of F2 rats, non-ischemic LVs from F2 rats were isolated after CF measurements and stored at −80 °C for gene expression analysis. A total of 118 RNA samples were processed in 12 batches using the Maxwell 16 System RNA Purification Kit (Southampton Science Park, Southampton, UK). In each sample, 200 μL of lysate was added for every 25 μg of tissue, and the samples were homogenized until no solid sample could be seen. 200 μL of sample lysates were then diluted in 400 μL of RNA dilution buffer and vortexed to ensure complete dissolution. Add 50 μL of clarifying agent to sample lysates to bind DNA at 70°C. The clarifier-sample mixture was then cooled and centrifuged at 12,000 rpm for 2 minutes. Place the bottom phase in a Maxwell robot to obtain a final 40 μL RNA sample in RNase-free water. RNA purity was then assessed using NanoDrop. RNA integrity was assessed using the Agilent 2100 Bioanalyzer platform, and samples with RNA integrity values <8 were discarded.

Expression was measured using Affymetrix Rat GeneChip 1.0ST. Raw probe intensities were then background corrected by subtracting the average of background probes with the same GC content. A VSN normalization step (Huber et al.) was then applied to the data, and median smoothing was used to summarize probeset expression for each transcript.

genotype data

DNA from the F2 population was extracted from tail tissue samples using robotic DNA extraction on a Maxwell 16 system. DNA quantity and quality were assessed using NanoDropTM and Invitrogen's Quant-it assay. High-throughput genotyping was performed on custom genotyping bead chips using Illumina's GolgenGate assay. 16,543 BN-SHR SNPs were obtained from the STAR consortium (http://rgd.mcw.edu/), and the 160 bp sequence surrounding each SNP was retrieved based on the SHR genome sequence (Atanur et al., GenomeRes, 20:791-803 (2010) ). SNPs and their surrounding sequences were submitted to Illumina for bioinformatics evaluation and attributed to quality scoring. Then 768 SNPs that were uniformly distributed on the genome and had a quality score of 0.95 or higher were selected to constitute the bead chip. After hybridization and imaging, genotypes were called using GenomeStudio software and the GenCall algorithm. Parental and F1 samples were added to F2 samples as a control for genotyping quality (parent-parent-progeny error/P-P-C); sample replicates were also placed on bead chips to estimate reproducibility error. SNPs with poor cluster separation, reduced mean normalized intensity (R<0.2) or low MAF (MAF<0.1) were excluded. SNPs showing significant deviation from Hardy Weinberg equilibrium (P<10-3) were also excluded. After genotype filtering, the P-P-C precision was about 97%, and the replication error rate was estimated to be about 0.01%. The overall call rate was 93%. Missing genotypes were imputed using fastPhase with default settings and two founder haplotypes.

coronary flow mapping

Coronary blood flow QTL was mapped using the matlab application of HESS. All 6 coronary flow phenotypes (mean and maximum, 3 time points) were included in the model as dependent variables. Markers in high LD (r2>XX) were removed prior to analysis, and SNP genotypes were used as regressors in the model. The default parameters are used for Hierarchical Evolutionary Stochastic Search (HESS). A marginal posterior probability of 0.8 is required to identify genetic modulation of phenotypes by SNPs. By taking the range between two markers around the marker selected by HESS, the corresponding haplotype is obtained.

eQTL mapping

eQTLs were mapped using the ESS++ program. Three mRNA expression levels measured by microarrays in F2 were included in the model as dependent variables, and genotype was used as a regressor.

result

This study identifies WARS2 as a novel gene for coronary blood flow and capillary density. The genetic control of coronary blood flow is unknown. Described herein is the use of methods similar to those described in McDermott-Roe et al., Nature, 478:114-118 (2011); Petretto, et al., Nat Genet, 40:546-552 (2008) in rats. A study of it in scale F2 interaction experiments.

Coronary blood flow was measured at three time points in 172 rats in the Brown Norway-spontaneously hypertensive rat (BN-SHR) F2 cohort using a Langendorff preparation. The role of widely accepted determinants of coronary blood flow in rat populations was investigated. While a strong effect of left ventricular relaxation pressure ((dp/dt)min) on coronary flow was found (r=-0.34, p=4.6×10 ^-6 ), no effect was observed for systolic or diastolic blood pressure (p =0.19 and p=0.36), suggesting a blood pressure-independent mechanism regulating coronary blood flow in the rat population.

Maximum and mean coronary blood flow were plotted at three different time points using HESS (Bottolo et al., Genetics, 189:1449-1459 (2011)). By exploiting the correlation structure between these phenotypes, HESS jointly simulates multiple phenotypes.

A strong and consistent association of coronary blood flow measured at basal, post-ischemic, and infarct levels with a single locus located on rat chromosome 2q34 was observed (shown in Figure 1). The upper limit of haplotype restriction was derived from the position of two adjacent markers, suggesting that the causal genetic variant could be located between 189.3 and 194.5 Mb.

RNA-seq analysis in left ventricular tissue from BN and SHR identified 42 expressed genes in this region, three of which had significant eQTLs (Fig. 1). Two genes Wars2 and Cathepsin-S (Cathepsin-S) were found to have non-synonymous variants. Cathepsin S has variations at partially conserved protein sites and is low expressed in the heart. Wars2, on the other hand, is a mitochondrial aminoacyl tRNA synthetase (aARS) highly expressed in the heart and is nuclear-encoded and mitochondrial localized; it transfers tryptophan to its cognate tRNA.

Many aARSs (tyrosine ARS (YARS), serine ARS (SARS), cytoplasmic tryptophan ARS (WARS)) acquired atypical functions in angiogenesis during the course of evolution, and cleavage products of WARS have been shown to inhibit VEGF signaling. Therefore, Wars2 was prioritized as a candidate gene controlling coronary blood flow at the rat 2q34 locus.

The resistance of small capillaries greatly controls blood flow in the heart. The capillary density of the loci was examined in crosses and in F2 cross animals conditioned on genotype, and a significant effect of the loci was found (P = 1.3 x 10 ^-13 ) (see Figure 2).

Example 2

Characterization of WARS2 and its interaction with WARS

Materials and Methods

Anti-L13a and Annexin A were from Cell Signaling. Alexa-488/568 goat anti-mouse/rabbit IgG was from Molecular Probes. Anti-VEGF and GAPDH were from Santa Cruz, all other antibodies were from Abcam. Polyclonal anti-WARS2 rabbit antisera were obtained after immunization of rabbits with KMSKSD PDK LAT VC (SEQ ID NO: 15) or CIL TSM KKV KSL RDP S (SEQ ID NO: 16) peptides. They were produced using the corresponding peptides from Eurogentec (England) and purified chromatographically. The L53F mutant WARS2 cDNA was generated using the Quikchange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions using a plasmid construct (Origene) containing the human WARS2 cDNA with C-terminal myc-DDK or GFP as a template. L53F was verified by nucleotide sequencing. Adenoviruses (Ad-GFP, Ad-WARS2) were purchased from SignaGen Laboratories. Human WARS cDNA was synthesized from RNA extracted from human HUVEC cells, and the coding region was PCR amplified using primers containing EcoRI and Kosac sequences in the forward primer and flag sequence and XbaI in the reverse primer. After sequence verification, WARS was cloned into pLVX-EF1a-IRES-Puro (Clontec).

Cell Culture, Transfection, and Adenoviral Transduction

Human embryonic kidney (HEK) 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Human umbilical vein endothelial (HUVEC) cells and media (EBM single Quats and EBM-2 single Quats) were purchased from Lonza and used before passage 7. For plasmid transfection (for HEK293), cells were seeded one day before 70% confluence and lipofectamine 200 (InvitroGene) transfection reagent was used according to the company's instructions. For HUVEC transfection, Polyplus jetPEI-HUVEC transfection reagent (POLYPLUS-TRANSFECTION Inc. New York) was used according to the company's instructions. For gene transfer, grow HUVECs to 80% confluency in 6-well plates or T75 flasks. Cells were cultured at a multiplicity of infection (MOI) of 200 plaque-forming units (pfu) in HUVEC growth medium in a total volume of 1 ml (6-well plate) or 10 ml (T75 flask). divert. 24 hours post-infection, cells were incubated in medium containing 1% FBS for 1 hour and then supplemented with 500 U/ml human recombinant IFN (R&D Systems) for an additional 24 hours before processing for analysis.

GST fusion protein for enzyme assay

WARS2 and WARS2(L53F) cDNAs were cloned into commercially available vectors (see table), expressed using IPTG induction and purified using standard sequencing based on GSH beads. Purified protein was quantified using a standard malachite green assay (Cayman chemicals).

RNA extraction from cultured cells

Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Briefly, cells were lysed in 350 μl RLT buffer and applied to a Shedder column (Qiagen) and spun at 14,000 rpm for 1 min. The lysate was mixed with an equal volume of 70% ethanol and applied to an RNeasy column, which was then centrifuged and washed with the provided RW1 and RPE buffers. Finally, total RNA was eluted in a volume of 30 μl of RNase-free water and quantified by measuring absorbance at 260 nm using a Nanodrop 1000 (Thermo Fisher Scientific). Store RNA at -80 °C until needed.

Quantitative real-time PCR (QPCR) analysis and WARS splicing PCR

Total RNA (1 μg) was reverse transcribed using the iScript First Strand cDNA Synthesis Protocol (Bio-Rad). The resulting cDNA (1 μl) was subjected to QPCR in 20 μl reactions using a SYBR GreenJumpStart Taq ReadyMix (S4438, Sigma) and run on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Three independent 6-well cultures per siRNA treatment were used in at least three independent experiments per set, and each sample was run in duplicate. All data were analyzed using the 2-ΔΔCT method.

Table 1. Primer sequences

To detect WARS splice variants, 1 μl of cDNA from WARS and 1/20 dilution of 28S were used in a 50 μl PCR reaction and 24 cycles (determined from the melting curve in QPCR) were used.

Protein extraction and Western blotting

Cells were lysed in Cell Lysis Buffer (9803, Cell Signaling) containing protease inhibitors (Sigma) and phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific) and PMSF (0.5 mM, Active Motif). Cell lysates were then briefly sonicated and centrifuged at 14,000g (4°C) for 10 minutes. The supernatant was retained and the protein concentration was determined using the bicaprylic acid assay with bovine serum albumin standards. Protein samples (20 μg) were separated on 10% or 13% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies as described in the legend. Immunoreactive bands were visualized with horseradish peroxidase-conjugated secondary antibodies using peroxide and LumiGlo reagent (Cell Signaling).

siRNA-mediated knockdown of WARS2

HUVEC cells were seeded in 6-well plates. After 24 hours, cells were transfected with WARS2-targeted ON-TARGETplus SMARTpool using DharmaFECT-1 (Thermo Scientific) according to the manufacturer's instructions. For a given transfection, 24 μl DharmaFECT1 and 476 μl opti-MEM (Invitrogen) were mixed in one tube, while 5 μl of the above WARS2-targeted or scrambled (scrambled) control siRNA (20 μM) was mixed with 490 μl opti-MEM in Mix in the second tube. After 5 minutes of incubation at room temperature, the above solutions were mixed and left at room temperature for an additional 20 minutes before being added to the cells. The final siRNA concentration was 25 nM. Knockdown efficacy was assessed by QPCR 2 days after transfection and found to be at least 85%. Although the two anti-WARS2 antibodies we prepared could detect WARS2 in lysates from cells expressing WARS2-flag, knockdown efficacy could not be determined by western blotting because no antibody against WARS2 could detect endogenous WARS2.

Immunofluorescence

Cells were seeded onto sterile coverslips at approximately 80% confluency. For immunofluorescence analysis, cells were fixed in 3% (w/v) paraformaldehyde for 15 min at room temperature, permeabilized in phosphate-buffered saline (PBS) containing 0.3% TritonX100 for 4 min, and incubated with 1 % (w/v) bovine serum albumin (bovine serum albumin, BSA) in PBS for blocking. Cells were incubated with the primary antibody for 1 hour (diluted 1/40-100), then incubated with Alexa 488/568-conjugated goat anti-mouse/rabbit IgG (highly cross-absorbed; diluted 1/100) for 1 hour, both Performed at ambient temperature. Coverslips were mounted on glass slides with Vectashield antifade mounting medium (Vector Laboratories Inc.) containing DAPI. Cells were observed on a Leica confocal microscope using a 60x oil objective, and images were analyzed using Leica confocal software (Leica Microsystems (UK) Ltd, Milton Keynes, UK).

Preparation of L53F and wild-type WARS2 and WARS lentiviruses

WARS2 (WT and L53F) or WARS-flag cDNA tagged with flag or GFP was subcloned into pLVX-EF1α-IRES-zs Green (cat# 631982, Clontec) or pLVX-EF1α-IRES using EcoRI/XbaI sites - in mCherry (Cat. No. 631987, Clontec) or pLVX-EF1α-IRES-Puro vector (Cat. No. 632183, Clontec). Specific viruses were produced in Lenti-X 293T cells using the Lenti-X HTX Packaging System (631247) according to the manufacturer's protocol (Clontec).

Primers used to generate Wars cDNA

F: gggaattcgccgccgcgatcgcCAAAGGATGAAATTGATTCTGCAGT (SEQ ID NO: 25) (EcoRI, Kosac)

R: GctctagattacttatcgtcgtcatccttgtaatcCTGAAAGTCGAAGGACAGCTT (SEQ ID NO: 26) (XbaI, flag).

Primers used to generate Wars2-FLAG

Forward: GGGAATTCGTCGACTGGATCC (SEQ ID NO: 27)

Reverse: GcteagaGCCGGCCGTTTAAAACCTTATCG (SEQ ID NO: 28) (XbaI)

Primers used to generate Wars2-GFP

Forward: GGGAATTCGTCGACTGGATCC (SEQ ID NO: 29)

Reverse: 5'-GCtctagaGCCGGCCGTTTAAACTCTTTCT-3' (SEQ ID NO: 30) (XbaI)

Mitochondrial fractionation and exosome secretion

To isolate cytoplasm and mitochondria, the Mitochondria Fractionation Kit (ActiveMotif) was used according to the instruction manual. The purity of the mitochondrial and cytosolic fractions was verified using antibodies against the mitochondrial protein complex I (Mitoscience) or the soluble cytosolic protein tubulin (Sigma). Exosome pools were generated from HUVEC lysates using a kit (Invitrogen, 4478359) according to the manufacturer's instructions.

Immunoprecipitation and mass spectrometry

For immunoprecipitation, cell lysates were prepared in lysis buffer (cell singaling) containing protease inhibitors and phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific) and PMSF (0.5 mM). Anti-FLAG M2 affinity gels (Sigma) were used to purify Wars2flag-tagged proteins according to the company's protocol. Before elution, the protein beads were washed with 0.5% NP40 in PBS, then three times in 50 mM ammonium bicarbonate, and the binding was eluted in 200 μg/mL FLAG peptide (3xflag peptide, Sigma) in 50 mM ammonium bicarbonate. of protein. Mass spectrometry was performed using full-solution digestion with full-qualitative-plus-quantitative analysis.

result

WARS2 and WARS2(L53F) localize to mitochondria in HEK cells

WARS2 is a nuclear-encoded, mitochondrial-localized aARS. The localization of human WARS2 and mutated human WARS2(L53F), which has the same amino acid changes as in rat, was examined. Both mutant and wild type localize to mitochondria (shown here in Figure 3).

WARS2 binds to WARS, but mutant WARS2(L53F) does not

aARS can form higher orders of oligomerization with themselves and each other that affect their function. tested whether WARS2 binds to its cytoplasmic counterpart WARS, which has known anti-angiogenic functions. WARS2 was found to associate with WARS in HUVEC cells. This effect was reduced by the L53F mutation (as shown in Figure 4).

WARS2 expression affects WARS secretion, an effect mediated at the protein level

WARS fragments have anti-angiogenic activity by binding to VE-cadherin. Regulation of WARS secretion is unclear. WARS2 overexpression inhibits WARS secretion, whereas WARS2 gene silencing increases WARS secretion. This effect is mediated at the protein level and was not found to be associated with splicing or transcriptional effects.

HUVEC cells were cultured and infected with adenovirus encoding WARS2 or transfected with siRNA against WARS2. Control virus and siRNA were used in all experiments. WARS2 gain-of-function reduces WARS secretion compared to controls. Loss of function of WARS2 increased WARS secretion compared to controls (see Figure 5). Taken together, these data show that WARS2 regulates WARS secretion that would have downstream effects on angiogenesis. Although WARS transcription is known to be increased by IFN-γ stimulation and there is alternative splicing of WARS, the contribution of WARS2 to these events is not shown here (see Figure 6A). Therefore, the effect of WARS2 on WARS is at the protein level.

Cellular localization of WARS and WARS2 interactions

WARS is predominantly a cytosolic protein, but components of WARS are mitochondrial localized, and this subpool increases following interferon (IFN)-γ stimulation in HUVECs.

This paper demonstrates the interaction between WARS and WARS2. In subcellular fractionation experiments, WARS was detected in mitochondria, and this subpool increased upon IFN-γ stimulation. Therefore, this protein-protein interaction is expected to occur in the mitochondrial fraction, and this mitochondrial fraction may play a role in the secretion of WARS from cells (see FIG. 6B ).

WARS2 interacts with components of the GAIT complex

To further understand the protein-protein interactions of WARS2, mass spectrometry (MS) was performed. In the experiments herein, MS data indicated that WARS2 interacts with EPRS (an aARS), GAPDH and RPL14a. Together with NSAP1, these proteins define the GAIT complex, which is important for controlling protein expression of VEGF and inflammatory genes by binding to the 3'UTR of target genes.

MS data alone are inconclusive as many interactions are identified in this type of experiment and downstream confirmation is required. To confirm this interaction, co-IP experiments were performed with WARS2 and the WARS2(L53F) mutant. Interactions of WARS2 (but not the L53F mutant) with EPRS, GAPDH and RPL14a were documented (shown in Figure 7).

Furthermore, as shown here, overexpression of WARS2 caused a significant loss of RPL14a by targeting RPL14a to the proteasome, an effect that was reversed by the proteasome inhibitor MG132 (cf. Figure 8).

These data show that WARS2 functions at a second level in the control of VEGF activity, here by modulating the activity of the GAIT complex, which in addition to controlling many cytokines and chemokines (e.g., CXCL13, CCL22, CCL8 In addition to the translation of CCR3), it also controls the translation of VEGF.

Atypical secretion of WARS via the exosome/microvesicle pathway

Previous studies have shown that WARS is secreted, but the secretion pathway is atypical and not characterized. The effect of brefeldin A, an agent that inhibits Golgi-mediated secretion, on WARS secretion was examined in HUVEC cells. As previously stated, no effect was observed. Further studies showed that WARS was secreted in exosomes, which was inhibited by WARS2 (Figure 9). This defines a new secretion pathway for WARS in exosomes.

Example 3

Loss of function of WARS2 in zebrafish leads to impaired angiogenesis and impaired cardiac function

method

Morpholino experiments in zebrafish and imaging of zebrafish vascularization were performed using standard methods.

result

To examine the in vivo effects of Wars2 on angiogenesis and the heart, Wars2 activity in zebrafish was inhibited using a morpholino targeting the first codon of Wars2. There were significant effects on fish morphology and survival due to defects in vascularization and cardiac function, summarized in Figures 10-12.

Example 4

siWARS2 knockdown in human embryonic kidney (HEK) 293 cells

method

HEK cells were seeded on 10 cm dishes one day before transfection. Transfection was performed using RNAimax (manufacturer's protocol) at a density of approximately 50%. siRNA was prepared by using a mixture of 120 nM siRNA and 20 μl lipofectamin RNAimax in 1 ml OPTIMEM. Cells were incubated, washed 1X with PBS, then covered with 5ml OPTIMEM, and lipofectamin/siRNA mixture was added dropwise. Cells were incubated (6 hours) and then the medium was changed to (DMEM/10% FBS). RNA was extracted according to the protocol (Qiagen). RNA quantification was assessed using a Nanodrop and quality was determined based on the OD ratio 260nm/280um. Reverse transcription was performed using 1 μg of RNA (Biorad iScript™ cDNA synthesis protocol) and QPCR was performed using the QuantiFASTSYBR green protocol.

Primers for human WARS2

F GCCACCGTCCGAATAACAGA (SEQ ID NO: 31)

R CATGCACCGCCACTATGTTG (SEQ ID NO: 32)

Primers for human GAPDH

F GGAGTCAACGGATTTGGTCG (SEQ ID NO: 33)

R ATCGCCCCACTTGATTTTGG (SEQ ID NO: 34)

Loop condition:

95C 5 minutes

95C 10 sec]

60C 30 sec] 40x

95C 15 seconds

60C 1 minute

All samples were analyzed in duplicate and mean and standard deviation determined. The specificity of amplification was determined by melting curves in the range 60-95°C. Normalization to GAPDH was performed using the standard ddCt method.

The experiment was repeated 5 times, and the following results are shown in Table 2 (and Figure 15).

Table 2. Results of siWAR2 knockdown in HEK 293

experiment 1 2 3 4 5 average standard deviation non-targeting control 1 1 1 1 1 1 0 siRNA 1 0.26 0.13 0.18 0.13 0.14 0.17 0.05 siRNA 2 0.47 0.14 0.13 0.11 0.09 0.19 0.14 siRNA 3 0.43 0.22 0.43 0.37 0.30 0.35 0.08

WARS2 siRNA1:

Sense: 5'-GCAUUCAACCUACAGGAAU-3' (SEQ ID NO: 50)

Antisense: 5'-AUUCCUGUAGGUUGAAUGC-3' (SEQ ID NO: 51)

WARS2 siRNA2:

Sense: 5'-CCGACAUUCUGUUGUACAA-3' (SEQ ID NO: 52)

Antisense: 5'-UUGUACAACAGAAUGUCGG-3' (SEQ ID NO: 53)

WARS2 siRNA3:

Sense: 5'-GCUGGACAAGGACCAUUUA-3' (SEQ ID NO: 54)

Antisense: 5'-UAAAUGGUCCUUGUCCAGC-3' (SEQ ID NO: 55)

These three siRNA sequence pairs target regions of the WARS2 sequence described below, respectively. In some aspects, the siRNA sequence comprises an additional thymine (T) at the 3' end of both the sense and antisense strands of the siRNA pair to increase the resistance of the siRNA to intracellular nuclease activity. In a specific aspect, the sense and antisense strands of the siRNA comprise two Ts (TT) at the 3' ends.

siRNA1 target GCATTCAACCTACAGGAAT (SEQ ID NO: 35)

siRNA2 target CCGACATTCTGTTGTACAA (SEQ ID NO: 36)

siRNA3 target GCTGGACAAGGACCATTTA (SEQ ID NO: 37)

Figure 14 shows the cDNA of WARS2 indicating where the three siRNAs target, as described above.

Example 5

WARS2 SNPs associated with higher WARS2 expression control WARS levels in human plasma

WARS ELISA

WARS in human plasma was determined using ELISA and compared with the SNP genotype of WARS2 (rs984222). WARS levels in plasma were determined using ELISA according to the manufacturer's instructions (ABIN366223; Antibodies-online GmbH, Aachen, Germany). Plasma was collected from healthy volunteers, aliquoted and stored at -70°C. 100 μl of undiluted plasma was used for each measurement. A standard curve was performed in duplicate and a detection limit of 0.078 ng/ml was established. OD at 450 nm (corrected at 540 nm) was read on a microplate reader (uQuant; Biotek Instruments, UK).

WARS2 SNP determination

SNP ID: rs984222 was used in the WARS2 SNP assay.

Assay: Life Technologies Assay ID: C__8699051_10

System used: Life Technologies Stepone plus rtPCR

Table 3. PCR conditions:

Fluorescence sequence:

TTCATATTCTGTCGAGACACCCATC[C/G]CCCTGTGTTTCACTTGTCTGATTAC (SEQ ID NO: 38)

VIC dye C nucleotide, FAM dye G nucleotide

This study explored the WARS2 SNP RS984222, which is polymorphic with C/G transversion substitutions. To study the rate of this polymorphism in a group of healthy volunteers, a premade assay from Life Technologies (Assay ID: C__8699051_10) was used. The assay uses a VIC/FAM dye mixture to determine the presence of SNPs. The assay was performed on real-time PCR using a LifeTechnologies Stepone Plus and analyzed with Stepone Plus software v2.1, which generates allelic discrimination maps to determine SNPs.

result

The data show that WARS2 SNPs that control WARS2 expression also control WARS levels in human plasma. As shown herein, increased expression of WARS2 reduces WARS secretion. Using a sandwich ELISA approach (see Methods section above), it was determined whether the WARS2 SNP rs984222, which is associated with higher levels of WARS2 expression in various tissues, including adipose, was associated with reduced circulating levels of WARS in human plasma. This reveals the role of SNPs on WARS in human plasma. Individuals homozygous for the G:G allele (moderate WARS2 expression in tissues) compared to the G:C allele (moderate WARS2 expression in High WARS2) with lower levels of WARS in human plasma. See Figure 16.

Example 6

WARS2 expression regulates WARS secretion in exosomes

method

Cell Culture and Immunocytochemistry

Human umbilical vein endothelial cells (HUVEC; Lonza) were cultured using EBM-2 medium (Lonza). After two passages, cells were digested with TrypLETM (Life Tech) and seeded into 8-well chamber slides (ibidi) at a density of 5,000 cells/ ^cm2 . At 50% confluency, adenovirus (PRECISION TECHNOLOGIES) expressing human WARS2 or GFP was added at a multiplicity of infection of 100 (48 hours). Low serum EBM-2 medium was used, and selected cells were stimulated with interferon-γ (500 units/ml, 48 hours). Cells were fixed with 4% formaldehyde for 15 min and washed three times with PBS. Cells were treated with 0.1% Triton-100 (10 minutes) and washed three times with PBS. Blocking (30 minutes) was performed using 1% BSA in PBS. Primary antibody (WARS, Abcam, 1:200 in 1% BSA) was used and cells were incubated overnight at 4°C. Cells were then washed (x3) in PBS and incubated (30 min) with secondary antibody (Alex Fluor conjugated, Life Tech, 1:1000). After washing (3x) with PBS, cells were incubated with phalloidin (Alex Fluor conjugated, Life Tech, 1:200) for 30 minutes. Cells were then washed with PBS (x3), incubated with DAPI (Life Tech, 1:1000, 5 minutes), and treated with Gold Antifade (LifeTech) coverslips. Images were acquired using a laser scanning microscope (LSM 710, Zeiss) using the same settings.

result

Confocal images showing WARS secretion in exosomes. Furthermore, expression of WARS2 inhibited the secretion of these exosomes by trapping WARS in cells (see FIG. 17 ). HUVEC cells were infected with control virus (shown in Figure 17, left panel) or virus expressing WARS2 (shown in Figure 17, right panel), stimulated with interferon gamma, and then stained for WARS, actin and nuclei. In control cells, membrane-bound exosomal microvesicles containing WARS were seen budding from the plasma membrane of the cells (arrows). In contrast, in WARS2-infected cells, a large amount of WARS accumulated on the plasma membrane (*), but not in exosomes.

Example 7

Disruption of WARS2 by zinc finger nucleases leads to reduced vessel density

method

Disruption of Wars2 in rats

The BN rat Wars2 locus in the terminal region of the first exon was targeted with a zinc finger nuclease (ZFN) directed against the ZFN target site:

ACCCACAGCTACTGCggctcCCCAGGTAACCCGAG (SEQ ID NO: 39) (Sage laboratories, PA, USA). Gene disruption was screened by sequencing and an 8 bp deletion was identified in exon 1 of the ZFN target site. This results in a frame shift after amino acid 27 of the Wars2 protein and an early stop in exon 2 after amino acid 53 of Wars2.

Genotyping of mutant lines

Genomic DNA was extracted from toeclip samples using the E.Z.N.A tissue DNA kit (OMEGA bio-tek) according to the manufacturer's protocol. After purification, PCR was performed on each sample consisting of JumpStart Taq ReadyMix (Sigma) and 1 μM of the following primers:

(i) 5'-GTGAGTGCTGGCGCTTCATC (SEQ ID NO: 40) and

(ii) 5'-GGCCTAAAGCAGAAGGTCGG (SEQ ID NO: 41).

PCR cycling conditions: (i) 95°C for 5 minutes; (ii) 95°C for 30 seconds; (iii) 65°C for 30 seconds; (iv) 72°C for 30 seconds; (v) cycle 2 to 4 repeated 30 times; (vi) 72°C for 5 minutes; (vii) 4°C hold. PCR products were run on a 5% agarose gel. Expected band sizes are (i) wild type = 92bp (ii) heterozygous deletion = 92bp and 84bp (ii) homozygous deletion = 84bp.

result

These in vivo results in rats show that Wars2 levels affect capillary density and coronary blood flow. Specifically, when Wars2 levels were low, capillary density was low. Furthermore, when Wars2 was low, the capillary area in the heart was also low. Similarly, coronary blood flow in the heart, which is dependent on capillary density, was low when Wars2 levels were low. Figures 19A-C illustrate these results. Figure 18 shows efficient destruction of Wars2 using zinc finger nucleases.

Example 8

WARS2 activity inhibition

method

WARS and WARS2 AMP-glo enzyme assays

WARS and WARS2 enzyme assays were performed using an ATP depletion assay based on chemiluminescence optimized with AMP-Glo™ reagent (Promega) and using a 96-well format assay. The reaction buffer contained 25mM Tris-HCL pH7.2, 10mM MgCl ₂ , 50mM KCl, 2.5mM dithiothreitol, 0.1mg/mL bovine serum albumin, 0.2mM spermine, 10U/mL pyrophosphatase and the enzyme used The concentration is 200nM. Substrate concentrations were as follows: L-tryptophan 100 μM, bulk E. coli tRNA (Sigma) 200 μg/mL and ATP 100 μM. Reactions were performed at 37°C for 1 hour, followed by the addition of Reagent I (AMP-glo kit). Plates were incubated for an additional 1 hour at room temperature. At the end of this hour, AMP detection solution was added to all samples and incubated for 1 hour at room temperature. Data were collected by measuring luminescence with an Infinite M200 microplate reader (Tecan). Indolemycin was added at a concentration of 10 μM as indicated.

result

Suitable compounds can be used to inhibit WARS2 activity. For example, the present study shows that indomycin inhibits human mitochondrial WARS2, but not human cytoplasmic WARS activity. This inhibition of WARS2 activity can be combined with other modes of inhibition. For example, the WARS2 mutant rat mutation (L53F) was associated with lower vessel growth and lower WARS2 enzyme activity. Thus, using eg gene therapy approaches, the WARS2 mutant form L53F can be introduced into a subject to reduce enzymatic activity and thereby inhibit blood vessel growth. Such a targeted gene therapy approach could also be combined with other WARS2 inhibitors, such as indomycin or related compounds, to inhibit blood vessel growth. Figures 20A and 20B illustrate these results.

Example 9

In vivo knockdown of WARS2 in rat eyes using cholesterol-modified siRNA

method

Both non-targeting (NT) siRNA and siWars2 were purchased from Dharmacon and formulated in the provided sterile siRNA buffer. Fifty (50) μg of siWars2 was injected intravitreally into each eye of three F344 rats in a total volume of 5 μl. The same dose of NT control siRNA was injected into the contralateral eye of each animal. Retina and choroid were collected after 48 hours and total RNA was extracted by Trizol (Ambion). cDNA was then synthesized according to the manufacturer's protocol (BioRad).

Real-time PCR reactions were performed with SYBRgreen mastermix (Qiagen) to measure Wars2 expression by using the following primers and normalized to GAPDH mRNA levels:

Wars2 forward: 5'-TTTCCAGCAGTCTCAGGTGTC-3' (SEQ ID NO: 42);

Wars2 reverse: 5'TACAGGGTATGTGAGCAGGC-3' (SEQ ID NO: 43);

GAPDH forward: 5'-AAAGGGTCATCATCATCTCCGCC-3' (SEQ ID NO: 44);

GAPDH reverse: 5'-CCTTCCACGATGCCAAAGTT-3' (SEQ ID NO: 45).

result

Efficient knockdown of Wars2 in vivo in rat eyes using cholesterol-modified siRNA. Wars2-targeting siRNA showed stronger inhibition of Wars2 expression compared to non-targeting siRNA. See Figure 21.

NT-siRNA

Sense: 5'-UGGUUUACAUGUCGACUUU-3' (SEQ ID NO: 46)

Antisense: 5'-AGUCGACAUGUAAACCAUU-3' (SEQ ID NO: 47)

Wars2 siRNA

Sense: 5'-GCUCCAAUCAGAAGUGAUU-3' (SEQ ID NO: 48)

Antisense: 5'-UCACUUCUGAUUGGAGCUU-3' (SEQ ID NO: 49).

Example 10

An in vitro model of angiogenesis

method

Tube formation assay

48 hours after siRNA transfection or 48 hours after adenovirus transduction or siRNA gene knockdown, HUVECs were harvested and seeded in 96-well plates at a density of 8,000 cells per well with 50 μl of Matrigel (Corning Matrigel matrix, growth factor reduction) pre-coated. Images of each well (center position) were acquired with a 5X objective (DM3000 inverted microscope, Leica) after 8 hours of culture in complete EGM-2 medium. Images were analyzed by WimTube (Wimasis Image Analysis).

result

Modulation of WARS2 results in an increase and/or decrease in blood vessel formation (ie, angiogenesis). Loss of WARS2 function resulted in a very significant reduction in angiogenesis, as measured by tube formation by endothelial cells; total tube length; and number of branch points. Data can be seen in Figures 22A-C. In contrast, gain of function of WARS2 (shown in Figure 22D-F) resulted in a corresponding increase in these measures of angiogenesis.

Example 11

siWARS2 causes a decrease in endothelial cell proliferation

method

HUVEC cultured in 8-well chamber slides were fixed with 4% formaldehyde. Cells were permeabilized with 0.1% Triton x-100 and incubated with Alexa Fluor 488 phalloidin (Molecular Probes, 1:200 in PBS, 30 minutes). Nuclei were stained with DAPI (Molecular Probes, 1:1000 in PBS, 5 minutes) and cells were mounted with ProLong gold antifade mounting medium (Molecular Probes) or VECTASHIELD mounting medium (Vector Laboratories). Images were acquired using a confocal laser scanning microscope (LSM 710, Zeiss) and a super-resolution structured illumination microscope (ELYRA PS.1, Zeiss). Super-resolution images were post-processed by ZEN software (Zeiss) with SIM module according to the manufacturer's instructions.

Cell counts: Adherent HUVECs were harvested by trypsinization and counted using an automated cell counter (Countess Automated Cell Counter; Invitrogen).

result

The effect on cell morphology can be seen in the high resolution micrograph of Figure 23A. In the presence of repressed WARS2, actin fibers disappeared entirely, and mitochondria formed clumps. This reduces the ability of endothelial cells to migrate and divide; there is incomplete nuclear separation and cells accumulate in G2/M phase (see Figure 24A and B). A downstream effect of these changes is decreased proliferation due to cell death. Significantly inhibited proliferative capacity can be seen in the graph shown in Figure 23B.

Example 12

Inhibition of Wars2 prevents vessel growth in a model of manual breast cancer angiogenesis

method

Angiogenesis Assay of Matrigel Plugs

Thaw Corning Matrigel membrane matrix (LDEV-free: 5 ml) (Fisher, cat. no. CB-40234A) on ice or in a 4°C freezer. While keeping the Matrigel on ice, add a reconstituted stock solution (0.5 mg/ml in sterile PBS) of recombinant rat VEGF 164 (R&D Systems, Cat# 564-RV-010/CF) to the Matrigel ) to a final concentration of 500 ng/ml. 500 [mu]l of cold VEGF/Matrigel was filled into a cooled 1 ml syringe and capped with a 23 or 27 gauge needle (with a cap on it). Keep the syringe on ice to keep it cool until injection.

Rats were anesthetized using isoflurane or ketamine mixture. The mammary fat pad (MFP) between the lowest and second lowest nipples was pinched, slightly lifted, and 500 μl of Matrigel was injected into the MFP. Matrigel cures immediately. Five days after injection, rats were euthanized and matrigel plugs were carefully excised from adipose tissue. Wrap the tissue in foil and immediately freeze in liquid nitrogen.

result

In vivo vessel growth is blocked in a model of breast cancer angiogenesis in the presence of repressed Wars2. Three rats with normal Wars2 and three rats carrying a Wars2(L53F) mutation (using zinc finger nuclease mutations as described herein, see also Figure 18) were studied. In rats with and expressing normal Wars2, growth of blood vessels into the VEGF matrigel plug was observed in each of the three rats examined. In contrast, in the three rats that had suppressed Wars2 by using mutations in zinc finger nucleases, little growth of blood vessels into the VEGF matrigel plug was seen. Matrigel plugs with and without regrowth can be seen in FIG. 25 . Thus, WARS2 inhibition is beneficial in diseases characterized by enhanced and/or undesired blood vessel growth (eg, cancer and various eye diseases).

Example 13

Evaluation of the effect of Wars2 siRNA on reducing lesion size and leakage in a rat model of laser-induced choroidal neovascularization (CNV)

method

Non-targeting siRNA (negative control), Wars2 siRNA (test agent) and anti-VEGF Ab (positive control) were administered intravitreally into the eyes of 6-8 week old brown Norwegian female rats according to the following design:

Day 1: Bilateral laser treatment to produce three (3) lesions per eye

Day 3: Bilateral intravitreal injection of negative control, positive control or test agent

Day 22: In vivo fluorescein angiography

Day 22: Eyes are enucleated and fixed for future histological analysis

Three (3) groups of five (5) rats were used, as indicated in Table 4.

Table 4. Laser-induced CNV experimental details

All animals were housed in groups of three in large cages maintained in ventilated racks under standard animal care conditions.

anaesthetization

Using 20 units of ketamine (100 mg/ml) and 100 units of xylazine (20 mg/ml), ketamine and xylazine were mixed using a U-100 syringe. Anesthesia mixture was applied by IP injection at 1 μl/g (body weight).

Laser application to generate CNV damage

Animals were dilated with 1% Cyclogyl solution and protected from light. After observable dilation, animals were sedated with ketamine/xylazine. The fundus of sedated animals was observed and recorded using a Micron III small animal ophthalmoscope (Phoenix Research). Laser processing was performed using a thermal laser connected via a Micron III custom laser accessory. A total of 3 lesions were set per eye using a wavelength of 520 nm. The resulting fundus images were recorded and evaluated to confirm that the laser had successfully passed through Bruch's membrane to generate bubbles.

intravitreal administration

Animals were anesthetized with ketamine/xylazine, and Cyclogel and/or tropicamide were topically administered to dilate the pupils. After sedation and dilation, a total volume of 5 μl per eye was injected into the vitreous at the pars plana using a Hamilton syringe and a 33 gauge needle.

NT-siRNA:

Sense: 5'-UGGUUUACAUGUCGACUUU-3' (SEQ ID NO: 46)

Antisense: 5'-AGUCGACAUGUAAACCAUU-3' (SEQ ID NO: 47)

Wars2 siRNA

Sense: 5'-GCUCCAAUCAGAAGUGAUU-3' (SEQ ID NO: 48)

Antisense: 5'-UCACUUCUGAUUGGAGCUU-3' (SEQ ID NO: 49)

fluorescein angiography

Animals were anesthetized with ketamine/xylazine before receiving an IP injection of 10% sodium fluorescein at 1 μl/g body weight. Fundus images were then captured as 8-bit TIFF files using a Micron III and an exciter/barrier filter for the 488nm target wavelength. Standard color fundus photographs were also captured for each eye.

Imaging and damage quantification

All TIFF images were quantified using computerized image analysis software (ImageJ, NIH, USA). Lesions were then tracked individually free-hand, quantifying the area in pixels, and color fundus photographs were used as a reference for lesion location. The avascularized area in the center of the injury was not included in the area calculation. If bleeding was present or two lesions overlapped, these lesions were excluded from the analysis.

organization collection

Animals were anesthetized with ketamine/xylazine (80/10 mg/kg) and then euthanized by IP administration of Euthasol (pentobarbital) at 200 mg/kg. After euthanasia, eyes were enucleated and individually fixed in 4% paraformaldehyde. After fixation, store all eyes in individual 2 mL screw cap polypropylene tubes. Store samples at room temperature.

Data and Statistical Analysis

Statistical analysis of significance was performed using Graphpad Prism software (version 4.0) using an unpaired t-test. Only changes with p-values <0.05 were considered statistically significant.

Example 14

Experimental results carried out in embodiment 13

Following laser treatment, choroidal neovascularization proceeded as expected in negative control animals (ie, those injected with non-targeting siRNA). However, in the case of the therapeutic intervention control (anti-VEGF antibody) and test agent (WARS2 siRNA), a significant reduction in the extent of neovascularization was observed.

Example 15

The effect of WARS2 knockdown on the number of human A549 lung cancer cells

method

The day before transfection, the A549 lung cancer cell line (ATCC catalog: CCL-185) was cultured in 96-well plates at 10 ⁴ cells/well. The next day, the cells were transfected with a non-targeting siRNA control (SN001-10D, 10 nmol, from Singapore Advanced Biologics Pte Ltd) or human WARS2 siRNA (5'-CCGACATTCTGTTGTACAA-3'-SEQ ID NO:36). Transfection was performed as described in the protocol used by Life Technologies for their Lipofectamine RNAiMAX method.

2, 4 and 5 days after transfection, use Cell number was analyzed by luminescent cell viability assay (Promega).

result

The present study demonstrates that reducing WARS2 expression using siRNA targeting WARS2 significantly reduces the number of lung cancer cells in human A549 lung cancer. As shown in Figure 26, non-targeting siRNA had no effect, while a significant reduction in cell number was observed only two days after siRNA transfection, and a 70% reduction in number by 5 days after transfection. Therefore, the proliferation of cancer cells was reduced or inhibited by reducing the expression of WARS2.

The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

While the invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that changes may be made in form and detail without departing from the scope of the invention as encompassed by the appended claims. kind of change.

Claims (73)

CLAIMS 1. A method of modulating angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that modulates WARS2 expression, WARS2 activity, or a combination thereof. 2. The method of claim 1, wherein the agent is a nucleic acid, a small organic molecule, an antibody, an aptamer, mutant WARS2, or a combination thereof. 3. The method of claim 1 or 2, wherein the agent inhibits WARS2 expression, WARS2 activity, or a combination thereof, thereby inhibiting angiogenesis. 4. The method of any one of claims 1 to 3, wherein the agent is a nucleic acid that binds a WARS2 nucleic acid. 5. The method of any one of claims 1 to 3, wherein the small organic molecule is indomycin or a derivative thereof. 6. The method of claim 4, wherein the nucleic acid is a small interfering ribonucleic acid (siRNA) and/or a morpholino oligomer specifically targeting WARS2. 7. The method of claim 1 or 2, wherein the agent enhances WARS2 expression, WARS2 activity, or a combination thereof, thereby enhancing angiogenesis. 8. The method of claim 1, 2 or 7, wherein the reagent is a vector directing expression of WARS2. 9. A method of inhibiting angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof. 10. The method of claim 9, wherein the agent is a nucleic acid, a small organic molecule, an antibody, an aptamer, mutant WARS2, or a combination thereof. 11. The method of claim 9 or 10, wherein the agent is a nucleic acid that binds a WARS2 nucleic acid. 12. The method of claim 10, wherein the small organic molecule is indomycin or a derivative thereof. 13. The method of any one of claims 9 to 11, wherein the nucleic acid is a small interfering ribonucleic acid (siRNA) and/or a morpholino oligomer specifically targeting WARS2. 14. The method of any one of claims 1-6 or 9-13, wherein the individual has retinopathy, macular degeneration, cancer, is obese, or a combination thereof. 15. The method of claim 14, wherein the retinopathy is diabetic retinopathy. 16. The method of claim 14, wherein the macular degeneration is age-related macular degeneration (AMD). 17. The method of claim 16, wherein the AMD is wet AMD or dry AMD. 18. A method of treating retinopathy in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof. 19. The method of claim 18, wherein the retinopathy is diabetic retinopathy. 20. A method of treating macular degeneration in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof. 21. The method of claim 20, wherein the macular degeneration is age-related macular degeneration (AMD). 22. The method of claim 21, wherein the AMD is wet AMD or dry AMD. 23. The method of any one of claims 18 to 22, wherein the agent is a nucleic acid, a small organic molecule, an antibody, an aptamer, mutant WARS2, or a combination thereof. 24. The method of any one of claims 18 to 23, wherein the agent is a nucleic acid that binds a WARS2 nucleic acid. 25. The method of claim 24, wherein the nucleic acid is a small interfering ribonucleic acid (siRNA) and/or a morpholino oligomer specifically targeting WARS2. 26. The method of claim 23, wherein the small organic molecule is indomycin or a derivative thereof. 27. A method of inhibiting cancer cell proliferation in an individual in need thereof comprising administering an effective amount of an agent that inhibits WARS2 expression, WARS2 activity, or a combination thereof. 28. The method of claim 27, wherein the agent is a nucleic acid, a small organic molecule, an antibody, an aptamer, mutant WARS2, or a combination thereof. 29. The method of claim 27 or 28, wherein the agent is a nucleic acid that binds a WARS2 nucleic acid. 30. The method of claim 28, wherein the small organic molecule is indomycin or a derivative thereof. 31. The method of any one of claims 27 to 30, wherein the nucleic acid is a small interfering ribonucleic acid (siRNA) and/or a morpholino oligomer specifically targeting WARS2. 32. The method of any one of claims 27 to 31, wherein the cancer cells are lung cancer cells. 33. A method of enhancing angiogenesis in an individual in need thereof comprising administering an effective amount of an agent that increases WARS2 expression, WARS2 activity, or a combination thereof. 34. The method of claim 33, wherein the agent is a nucleic acid, or a small organic molecule, an antibody, an aptamer, mutant WARS2, or a combination thereof. 35. The method of claim 33 or 34, wherein the agent is a vector directing expression of WARS2. 36. The method of any preceding claim, wherein the WARS2 activity comprises an interaction with WARS, EPRS, GAPDH, RPL14a, or a combination thereof. 37. A method for identifying the angiogenic potential of a tumor in an individual in need thereof, comprising determining one or more extracellular vesicles (EVs) obtained from said individual and/or their contents compared to a control WARS levels in an object, wherein an increase in the level of WARS in the one or more EVs and/or their contents compared to the control indicates that the tumor in the individual has low angiogenic potential and is consistent with the A reduction in the level of WARS in the one or more EVs and/or their contents compared to the control indicates that the tumor in the individual has high angiogenic potential. 38. The method of claim 37, wherein the control is the level of WARS in one or more EVs obtained from one or more tumor-free individuals. 39. The method of claim 37 or 38, further comprising obtaining a sample comprising the one or more EVs from the individual. 40. The method of claim 39, wherein the sample is biological fluid of the individual, biological tissue of the individual, or a combination thereof. 41. The method of claim 40, wherein the biological fluid is blood or plasma. 42. The method of any one of claims 37 to 41, further comprising obtaining one or more EVs or their contents from the individual. 43. The method of any one of claims 37 to 42, further comprising treating the individual based on the identified tumor angiogenic potential. 44. The method of claim 43, wherein if the level of WARS in the one or more EVs and/or their contents is reduced compared to a control, an effective amount of WARS2 expression, WARS2 activity, or The agents in combination treat the individual. 45. The method of any one of claims 37-44, wherein the one or more EVs are exosomes. 46. A method for identifying angiogenic potential in an individual with a cardiovascular disorder, comprising determining one or more extracellular vesicles (EV) and/or their contents obtained from said individual compared to a control WARS levels in said control, wherein an increase in the level of WARS in said one or more EVs and/or their contents indicates low angiogenic potential in said individual compared to said control, and compared to said control Relatively, a reduction in the level of WARS in the one or more EVs and/or their contents indicates high angiogenic potential in the individual. 47. The method of claim 46, wherein the control is the level of WARS in one or more EVs obtained from one or more individuals without a cardiovascular disorder. 48. The method of claim 46 or 47, further comprising obtaining a sample comprising the one or more EVs from the individual. 49. The method of claim 48, wherein the sample is biological fluid of the individual, biological tissue of the individual, or a combination thereof. 50. The method of claim 49, wherein the biological fluid is blood or plasma. 51. The method of any one of claims 46 to 50, further comprising obtaining one or more EVs or their contents from the individual. 52. The method of any one of claims 46 to 51, further comprising treating the individual based on the angiogenic potential of the identified cardiovascular disorder. 53. The method of claim 52, wherein if the level of WARS in the one or more EVs and/or their contents is increased compared to a control, an effective amount of enhanced WARS2 expression, WARS2 activity, or The agents in combination treat the individual. 54. The method of any one of claims 46-53, wherein the one or more EVs are exosomes. 55. A method of determining whether an individual in need thereof would benefit from anti-angiogenic therapy comprising: a) Genomic analysis of all or part of chromosome 1 of said individual, wherein said part comprises: TTCATATTCTGTCGAGACACCCATC[C/G]CCCTGTGTTTCACTTGTCTGATTA C (SEQ ID NO: 38); and b) detecting at least one allele at position 26 of SEQ ID NO: 38, wherein if at least one allele is G at position 26 of SEQ ID NO: 38, said individual will benefit from anti-angiogenic therapy. 56. The method of claim 55, wherein the individual is homozygous for G at the allele (G:G), heterozygous for G at the allele (G:C), or Homozygous for C at the allele (C:C). 57. The method of claim 55 or 56, wherein the portion of chromosome 1 comprises positions 119,503,593-119,504,093 of chromosome 1 . 58. The method of any one of claims 55 to 57, wherein detection of the at least one allele at position 26 of SEQ ID NO: 38 from the individual is compared to a control. 59. The method of claim 58, wherein the control is obtained from one or more individuals who responded to anti-angiogenic therapy, did not respond to anti-angiogenic therapy, or a combination thereof. 60. The method of any one of claims 55 to 59, wherein the individual has a tumor. 61. The method of any one of claims 55 to 60, further comprising obtaining a sample from the individual. 62. The method of any one of claims 61, wherein the sample is obtained from a biological fluid of the individual, a biological tissue of the individual, or a combination thereof. 63. The method of claim 62, wherein the biological fluid is blood or plasma. 64. The method of any one of claims 55 to 63, further comprising treating an individual having at least one allele that is G at position 26 of SEQ ID NO:38 with an anti-angiogenic therapy. 65. The method of claim 64, wherein the anti-angiogenic therapy comprises inhibition of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR), platelet-derived growth factor (PDGF), Tie-1 receptor (Tie -1R), Tie-2 receptor (Tie-2R), placental growth factor (PlGF), VE-cadherin, or combinations thereof. 66. The method of any one of the preceding claims, wherein the individual is a human. 67. A method of identifying an agent that modulates WARS expression, activity, or a combination thereof, comprising: a) determining WARS expression and/or activity in one or more extracellular vesicles (EVs) obtained from cells, tissues and/or organisms that have been contacted with the agent to be assessed; and b) comparing WARS expression, activity or a combination thereof as described in a) to a control, wherein said agent modulates WARS expression, activity, or combination thereof if WARS expression, activity, or combination thereof is altered in the presence of said agent as compared to said control. 68. The method of claim 67, wherein the control is WARS expression, activity, or a combination thereof of EVs in the absence of the agent. 69. The method of claim 67 or 68, wherein WARS expression, activity, or a combination thereof of the EV is reduced in the presence of the agent as compared to the control. 70. The method of claim 67 or 68, wherein WARS expression, activity, or a combination thereof of the EV is increased in the presence of the agent as compared to the control. 71. The method of any one of claims 67 to 70, further comprising contacting the cell, tissue and/or organism with an agent to be assessed. 72. The method of any one of claims 67 to 71, further comprising obtaining the one or more EVs from the cells, tissues and/or organisms that have been contacted with an agent to be assessed. 73. The method of any one of claims 67-72, wherein the one or more EVs are exosomes.